The present invention relates to adult tissue-derived connective tissue progenitor cells (CTPs) which exhibit high proliferation rate and multipotent differentiation potential which is maintained for at least 20 passages, and more particularly, to methods of generating and using such cells for cell based therapy and tissue engineering applications.
Cell-based tissue engineering is an evolving interdisciplinary area that offers new opportunities for clinical applications, creating a tool for repairing and replacing damaged or lost tissues with biological substitutes. The shortage of organ transplants and the exceeding number of patients on waiting lists greatly encourage the development of this field. The fundamentals of tissue engineering combine cells, bioactive matrices and chemically and biophysically defined in-vitro culture conditions. For tissue engineering, cells must be easily isolated, sufficient in numbers, with a great proliferation capacity and a well-defined differentiation potential. A number of cell sources have been suggested including primary cells and stem cells which are either host- or donor-derived. A wide array of matrices, either biologically or synthetically designed, are to provide the mechanical cues and three-dimensional environment, supporting cell attachment, migration, proliferation, differentiation and organization into complex tissues. Controlling stem cell proliferation and differentiation into any desired cell type requires the identification of chemicals (e.g., hormones and growth factors) and/or growth conditions (e.g., static or dynamic culturing conditions), which regulate the differentiation into the desired cell or tissue.
Connective tissue repair and regeneration are subjected to intensive research within clinical medicine. Damaged or disordered connective tissues, such as bone, cartilage and tendons need to be reconstructed or replaced due to traumatic injuries, degenerative diseases, tumor resections and congenital malformations. Current strategies in reconstructive orthopedic surgery include the use of autografts, allografts and artificial substitutes, all subjected to various limitations. While the use of cell grafts is limited by availability and morbidity, synthetic grafts are osteoconductively inferior to their biological counterparts, and could fail.
Thus, for cell based therapy and tissue engineering applications, methods of isolating and expanding stem or progenitor cells which can give rise to an unlimited amount of connective tissue cell lineages capable of forming connective tissue in vitro are highly desired.
Various studies attempted to identify culturing conditions which can be used to generate connective tissue progenitor cells which exhibit unlimited expansion in culture and differentiation potential to cells of the connective tissue lineages.
For example, bone marrow-derived MSCs can be cultured in a culture medium (e.g., DMEM) supplemented with serum. However, although cells cultured under such conditions and isolated from the first and second passage in culture were shown capable of differentiating into the adipocytic, chondrocytic, or osteocytic lineages (see e.g., Pittenger, M. F et al, 1999), the use of such cells in tissue engineering applications such as for the in vitro construction of a mature tissue was never demonstrated.
In another study Sottile, et al., 2002, cultured human trabecular bone-derived cells in a culture medium containing serum and fibroblast growth factor (FGF-2). However, the resulting cells reached confluency only after 10-20 days, demonstrating their relatively slow proliferation rate. Although cells isolated from the first passage could be induced to differentiate into osteoblasts, chondrocytes and adipocytes, their low proliferation rate limits their use in cell based therapy and tissue engineering applications.
In another study Zuk, P. A., et al., 2001, cultured processed lipoaspirate (PLA) cells which were obtained from fat tissues in a culture medium (DMEM) supplemented with serum. However, although the cells could be passaged 13 times, their proliferation rate was extremely slow as evidenced by the low passaging frequency (i.e., every 12-13 days). Thus, although PLA cells from the first passage were capable of differentiating into the adipogenic, chondrogenic, myogenic, and osteogenic lineages, their low proliferation rate limits their use for cell based and tissue engineering applications.
U.S. Pat. Appl. No. 20050260748 discloses a method of isolating adult stem cells from an adipose tissue and culturing them in a medium containing N-acetyl-L-cysteine, an antioxidant (e.g., vitamin C) and nicotinamide. For induction into osteogenic differentiation, the cells were cultured in a medium containing dexamethasone, L-ascorbate-2-phosphate and beta-glycerphosphate.
In yet another study, Mastrogiacomo, M., et al., 2005, cultured human skeletal muscle cells in a culture medium containing fibroblast growth factor (FGF-2) and dexamethason and following two passages in such a medium the cells were further induced to differentiate in vitro into the chondrogenic, osteogenic and adipogenic cell lineages. However, the potential of using muscle-derived stem cells cultured under such conditions for generating engineered tissues in vitro was never shown.
Thus, the currently available culturing methods of stem cells do not teach the in vitro construction an engineered tissue (e.g., a mature tissue) in the absence of a scaffold, carrier or a cell support. For example, none of the above-described methods enables the formation of a mature tendon tissue.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods of generating adult stem cells-derived multipotent cells which are suitable for tissue engineering devoid of the above limitations.